The present invention relates to a method for preparing a protein hydrolysate, more specifically to an enzymatically prepared protein hydrolysate.
In the food industry protein hydrolysates may be used as food products or as additives to food products.
Conventionally, protein hydrolysates are produced chemically by hydrolysing protein or proteinaceous material, such as for example defatted soy flour or wheat gluten, with hydrochloric acid under refluxing conditions. The resulting hydrolysates are cheap and tasty. However, chemical hydrolysis also results in the formation of chlorohydrins, such as monochlorodihydroxypropanols (MCDPs) and dichloropropanols (DCPs), the presence of which is undesirable in food products.
Alternatively, protein or proteinaceous material may be hydrolysed enzymatically. First, the relevant protein source is subjected to a (partial) hydrolysis with one or more suitable endoproteases. Then, the resulting protein fragments can be degraded completely or partly into the individual amino acids or dipeptides or tripeptides by the use of exopeptidases. Two categories of exopeptidases are available:
(i) amino-terminal exopeptidases (which start to cleave-off amino-acids, dipeptides or tripeptides from the protein fragment""s amino-terminal end);
(ii) carboxy-terminal exopeptidases (which start to cleave-off amino-acids or dipeptides from the protein fragment""s carboxy-terminal end).
Small peptide fragments (i.e. proteins extensively pre-hydrolysed with a suitable endoprotease) provide the basis for rapid generation of amino acids.
To achieve maximum amino acid generation, commercial exopeptidase-rich preparations such as Flavourzyme(copyright) (NOVO Nordisk, Denmark) and various Sumizyme(copyright) preparations (Shin Nihon, Japan), contain a mixture of various endoproteases to create as many starting-points as possible for the different exopeptidases. Furthermore, these preparations contain different types of amino-terminal and carboxy-terminal exopeptidases to overcome the relatively high specificity towards particular amino acids sequences that most exopeptidases possess. As a result of their complex nature, these currently available endoprotease/exopeptidase mixtures are expensive and determine to a large extent the cost of the resulting hydrolysate. As various endoproteases are readily available as relatively pure and cost effective products, it is the complex mixture of exoproteases, which is considered essential, that is the cost determining factor. For example, WO94/25580 describes the use of at least five proteolytic components to provide a protein hydrolysate which is useful as a flavouring agent.
The present invention relates to a method for preparing a protein hydrolysate. The method comprises contacting proteinaceous starting material, under aqueous conditions, with a proteolytic enzyme mixture which comprises only one exopeptidase. Cost-effective production of such single exopeptidase preparations may be feasible using well-documented cloning techniques.
In a preferred embodiment, the exopeptidase is a carboxypeptidase, more preferably a serine carboxypeptidase.
In another preferred embodiment, the exopeptidase is a heat-stable exopeptidase, more preferably a heat-stable aminopeptidase.
The present invention provides a method for preparing a protein hydrolysate which comprises contacting proteinaceous starting material, under aqueous conditions, with a proteolytic enzyme mixture which comprises only one exopeptidase. A proteolytic enzyme mixture according to the invention, i.e. comprising a single selected exopeptidase in combination with one or more endoproteases, may hydrolyse vegetable or animal protein at least as effectively as an enzyme mixture which contains several exoproteases. Since cost-effective production of such single exopeptidase preparations is feasible using well-documented cloning techniques, the method according to the invention enables the economic production of protein hydrolysates.
By only one exopeptidase is meant that at least 75% of the exopeptidase activity is derived from one enzyme. More preferably at least 90%, most preferably at least 95% of the exopeptidase activity is derived from one enzyme.
The exopeptidase preparations used in the present invention are preferably obtained using the cloned gene encoding the exopeptidase in a well producing gene expression system. Typically such expression systems provide for exopeptidase preparations in which at least 60%, preferably at least 75%, more preferably at least 85%, of the protein is represented by the expression product of the cloned gene.
Exopeptidases produced using these rDNA-techniques, that is cloning the gene encoding the exopeptidase in a host organism that overexpresses this gene, usually provide for enzyme preparations comprising less contaminating enzymatic activities and usually will not require costly recovery steps. Well known host organisms that overexpress cloned genes are fungi or bacteria for example Aspergillus, Trichoderma, E. coli, Bacillus etc.
Examples of protein or proteinaceous material which may be hydrolysed by the method of the invention are known to the person skilled in the art and include vegetable proteins such as soy protein, wheat gluten, rape seed protein, pea protein, alfalfa protein, sunflower protein, zein, and animal derived protein such as casein, egg white, whey protein and meat protein. As some vegetable proteins such as wheat gluten have low solubilities under the pH conditions used, chemically treated versions of such protein sources provide another interesting group of substrates. In a preferred embodiment of the invention, the proteinaceous material used is defatted soy flour or wheat gluten.
The method according to the invention may be used to prepare a hydrolysate which contains peptides, amino acids or both peptides and amino acids. These hydrolysates may be used conventionally, for example in several food applications, including applications in the bakery industry, e.g. for improving colour and flavour; the savoury industry, e.g. for the production of seasonings; the dairy industry, e.g. for cheese ripening; the meat industry, e.g. for taste enhancement, the brewery industry, e.g. for the production of a fermentable wort; the beverage industry, e.g. for supplementing beverages with amino acids or peptides. The person skilled in the art will understand that the method according the invention may be used in any application for which mixtures of several endoproteases and several exopeptidases are currently used to generate peptides or amino acids from proteins, be it to improve solubility, consistency, fermentability, liquefaction, colour, filterability, nutritional value, digestibility or flavour.
Either a carboxypeptidase or an aminopeptidase may be used as the single exoprotease present.
If a carboxypeptidase (CPD) is used, it is preferably a serine carboxypeptidase. Serine carboxypeptidases occur in fungi (preferred) and higher plants (K. Breddam, Carlsberg Res. Commun. Vol 51, p 83-128, 1986; S. J. Remington and K. Breddam, Methods Enzymol. 244: 231, 1994). Carboxypeptidase Y present in the vacuoles of baker""s yeast is a well-known representative of this category as well as the carboxypeptidases CPD-I and CPD-II, identified in Aspergillus niger (Dal Degan, F. et al, Appl. Environ. Microbiol, 58(7): 2144-2152, 1992), and CPD-S1 from Penicillium janthinellum (Breddam, K; Carlsberg Res. Commun. 53: 309-320, 1988). Particularly useful serine carboxypeptidases available from plants are carboxypeptidases MI, MII and MIII (K. Breddam et al., Carisberg Res. Comm. 52, 297 (1987). They are present in amongst others barley malt. Carboxypeptidase M from barley malt can easily be obtained in high yields from plants using the method described in WO91/14772.
The use of a serine carboxypeptidase as the single exopeptidase allows hydrolysis under acidic conditions at moderate temperatures. So when a single (only one) serine carboxy peptidase is used in hydrolysing the proteinaceous starting material, the single serine carboxy peptidase provides for at least 75%, preferably at least 90%, more preferably at least 95% of the exopeptidase activity during protein hydrolysis as measured under acidic conditions at moderate temperatures, such as a pH ranging from 4 to 6 and a temperature ranging from 45xc2x0 C. to 60xc2x0 C., preferably a pH of 4.5-5.5 at a temperature of 50xc2x0 C. to 57xc2x0 C.
If an aminopeptidase is used, it is preferably a heat-stable aminopeptidase. In this context xe2x80x98heat-stablexe2x80x99 is used to indicate that the enzyme""s temperature optimum is at a temperature of 60xc2x0 C. or higher. Heat-stable aminopeptidases have been described for bacteria such as Bacillus stearothermophilus, Talaromyces duponti and for Mucor sp. (Roncari, G et al Methods Enzymol. 45: 522, 1976). The obligate thermophilic bacterium Bacillus stearothermophilus contains aminopeptidase AP-II, a metal ion dependent, dimeric enzyme with identical sub units (Stoll, E. et al, Biochim. Biophys. Acta 438: 212-220, 1976). The pH optimum of this enzyme is between 7.5 and 8.0 and the temperature optimum well above 60xc2x0 C. The enzyme closely resembles aminopeptidase T (AP-T) from Thermus aquaticus. Even the N-terminal amino sequences of AP-II from Bacillus stearothermophilus and AP-T from Thermus aquaticus exhibit considerable similarity (Motoshima, H. et al; Agric. Biol. Chem., 54(9), 2385-2392, 1990). No industrial application has been suggested for these enzymes as yet.
The use of a heat-stable aminopeptidase allows hydrolysis under slightly alkaline conditions at a temperature of 60xc2x0 C. or higher, thus minimising the risk of microbial contamination during hydrolysis. So, when a single (only one) heat-stable aminopeptidase is used in hydrolysing the proteinaceous starting material, the single heat-stable amino peptidase provides for at least 75%, preferably at least 90%, more preferably at least 95% of the exopeptidase activity during protein hydrolysis, as measured under slightly alkaline conditions at a temperature of 60xc2x0 C. or higher, such as a pH ranging from 7 to 9 and a temperature of at least 60xc2x0 C., preferably a pH of 7.2 to 8.5 at a temperature of at least 65xc2x0 C.
Although the scientific literature has adequately described the broad spectrum properties of serine carboxypeptidases under acidic conditions and moderate temperatures, and similar properties of the aminopeptidases APII and AP-T under slightly alkaline conditions and temperatures well above 60xc2x0 C., industrial use of these exopeptidases to generate pools of free amino acids has not been described. This is surprising because either lengthy amino acid sequences or complete nucleotide sequences of the cloned corresponding genes for these enzymes are known. Therefore, overexpression and production in suitable production strains could be performed by the person skilled in the art using available recombinant DNA techniques.
Existing enzyme mixtures often contain contaminating enzymes such as e.g. glucosidases. In the method according to the invention substantially pure enzymes are used. This is particularly advantageous in applications where poor taste characteristics are generated by the uncontrolled breakdown of sugars, isoflavoids or saponins.
However, in certain cases the controlled breakdown of compounds other than proteins may be desirable. This may be achieved by adding substantially pure enzymes such as, amylases, glucanases, glutaminases, phytases, glycosidases, cellulases and pectinases to the proteases, or to the proteinaceous material before, after or together with the proteases. Consequently, the total enzyme mixture will depend on the specific application. For example, the enzyme mixture for preparing a flavouring agent, which is typically rich in glutamate, may include glutaminases for the conversion of glutamine (liberated during hydrolysis) into glutamate, in addition to one or more endoproteases and the single exopeptidase.